CN103525936A

CN103525936A - Specific identification for transgenic rice kefeng No.6 strain via adoption of RPA (Recombinase Ploymerase Amplification) technology

Info

Publication number: CN103525936A
Application number: CN201310506259.9A
Authority: CN
Inventors: 金芜军; 宛煜嵩; 徐潮; 李亮; 苗朝华; 黄卫红
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Biotechnology Research Institute of CAAS
Priority date: 2013-10-24
Filing date: 2013-10-24
Publication date: 2014-01-22
Anticipated expiration: 2033-10-24
Also published as: CN103525936B

Abstract

The invention firstly provides an RPA (Recombinase Ploymerase Amplification) detection method of identification of transgenic insect-resistant rice kefeng No.6 strain and in particular discloses a primer and probe combination used for indentifying transgenic insect-resistant rice kefeng No.6 via a recombinase ploymerase amplification technology; a sequence of a positive primer is expressed as SEQ ID No.1; a sequence of a negative primer is expressed as SEQ ID No. 2; a sequence of the probe is expressed as SEQ ID No.3. The invention also discloses a method for indentifying transgenic insect-resistant rice kefeng No.6. The method comprises the following steps: extracting DNA (Deoxyribose Nucleic Acid) of a sample to be detected as a template, performing RPA quick amplification and real-time fluorescence detection by using the primer; and proving that the detected sample contains components of the transgenic insect-resistant rice kefeng No.6 if an obvious amplification curve is obtained.

Description

Application RPA technology is identified rich No. 6 strain specificities of transgenic paddy rice section

Technical field

The invention belongs to biological technical field, be specifically related to apply recombinase polysaccharase isothermal amplification technique (Recombinase Ploymerase Amplification, RPA) and identify transgenic pest-resistant rice specificity method, and corresponding primer and probe combinations.

Background technology

DNA cloning is the main method of detection of nucleic acids at present, and conventional PCR detects needs accurate instrument and loaded down with trivial details testing sequence, is difficult to meet the requirement of Site Detection under non-lab environment.In recent years, nucleic acid constant-temperature amplification technology has obtained significant progress, and comparing nucleic acid constant-temperature amplification technology with normal PCR does not need expensive PCR instrument, and rapid amplifying goes out object fragment at short notice, has the advantages such as easy, quick, sensitive.RPA technology is DNA replication dna in simulation organism, based on the polymerase-mediated amplification principle of recombinase, develops, while utilizing recombinase and primer to form microfilament to search the sequence of complete complementary with it on template DNA, under the help of single-stranded DNA binding protein, template DNA is unwind, primer and template DNA start pairing formation and copy required 3 ' C-terminal freely, under the effect of archaeal dna polymerase, copy extension, forming new DNA complementary strand reaction product is also to increase with exponential.Different from conventional PCR reaction, RPA reacts required primer length and is generally 30-35nt.During design of primers for fear of form primer inner and between secondary structure, the increase of its length also makes design of primers and selects difficulty to increase, the design of primer sequence with select most important to the result of RPA.In RPA amplification system, add a fluorescently-labeled probe just can realize the Real-Time Monitoring of template amplification, each mark Yi Ge fluorescence group (FAM and BHQ1) in these two T bases in probe middle part, between Liang Ge group, there is an abasic site (dSpacer), this site can be identified from colibacillary exonuclease by a kind of, this enzyme has 3 '-5 ' 5 prime excision enzyme activity, can Shi Liangge fluorescence group separated, thus make the accumulation synchronised of fluorescent signal and amplified production.In conjunction with a portable amplified fluorescence detector, just can in 10-20 minute, fluorescence curve be detected.RPA technology has greatly shortened detection time, has simplified response procedures, combines make field detection become possibility with DNA rapid extraction technology, is with a wide range of applications.

The method that round pcr detects transgenic product has screening to detect, and gene specific detects, and builds specific detection and specificity of transformant and detects.Due to the uniqueness characteristic of foreign gene in acceptor gene group insertion point, therefore according to the connecting zone design RPA primer of external source insertion DNA sequence dna and rice genome, detect and there is very high specificity, can detect specifically this transformant and derivative strain thereof.

Transgenic pest-resistant rice section has entered the safety evaluation stage rich No. 6, has good Commercial Prospect.The at present not yet commercialization plantation of rich No. 6 of approval section of China, for preventing that transgenic paddy rice from entering production intermediate links without safety approval, in the urgent need to setting up fast and convenient detection method, for market surpervision and routine monitor.

At present, in the transgenic plant detection method of having reported, be mainly to utilize PCR instrument in laboratory, to carry out conventional detection, the method can't further meet the rapid detection of transgenic product.Also do not utilize at present RPA technology to identify the rich strain specificity of doing for No. 6 of transgenic paddy rice section both at home and abroad.

Summary of the invention

For the blank in above-mentioned field, the invention provides the RPA detection method of rich No. 6 strain specificities of accurate, quick, easy detection transgenic paddy rice section.

Technical scheme provided by the invention is: a kind of for identify the primer of rich No. 6 of transgenic pest-resistant rice section by recombinase polysaccharase isothermal amplification technique, its forward primer sequence is as shown in SEQ ID No.1, reverse primer sequence is as shown in SEQ ID No.2, and probe sequence is as shown in SEQ ID No.3.

The present invention also provides a kind of test kit of identifying rich No. 6 of transgenic pest-resistant rice section by recombinase polysaccharase isothermal amplification technique, and this test kit comprises above-mentioned primer and probe.

The present invention also provides a kind of method of identifying rich No. 6 of transgenic pest-resistant rice section by recombinase polysaccharase isothermal amplification technique: extract the DNA of testing sample as template, utilize the primer described in claim 1 to carry out fluorescence rapid detection, if obtain obvious amplification curve, prove rich No. 6 of sample Pin Wei transgenic pest-resistant rice section of institute.Implementation step is: in 50 μ L amplification systems of RPA amplification kit recommendation response, add each 2 μ L(10 μ mol/L of primer), probe 0.5 μ L(10 μ mol/L), template DNA 50ng.39 degrees Celsius of reactions of RPA augmentation detection instrument (or quantitative real time PCR Instrument) 20 minutes.

The inventive method is to design a large amount of RPA Auele Specific Primers according to the connecting zone of external source insertion DNA sequence dna and rice genome, therefrom filters out a set of primer and probe combinations that can effectively detect fast rich No. 6 compositions of transgenic paddy rice section.Utilize this to primer, to carry out fluorescence rapid detection, rich No. 6 genomic dnas of transgenic paddy rice section of take can obtain obvious amplification curve as template, but not the genomic dna of transgenic paddy rice bright extensive 63 and 4 kinds of rice materials of other transgenic paddy rice strains extensive No. 1 grades of China is that template amplification does not all have amplification curve.Template is diluted with water to 10000,2000,500,100,50 copies, result shows the target molecules that 100 copies can be detected, proves that the method has higher sensitivity, this is the inaccessiable sensitive level of prior art.The present invention provides the RPA detection method of rich No. 6 strain specificities of transgenic pest-resistant rice section first.The method is improved the ability of biological technology products aspect accurate, rapid detection.

Accompanying drawing explanation

The rich No. 6 specific detection figure of Tu1Wei section, wherein, 1: section's rich No. 6 (blades); 2: the rich No. 6 seed powder of section (5%); 3: the rich No. 6 seed powder of section (1%); 4: extensive No. 1 of China; 5: anti-excellent 97; 6: Kemingdao 1; 7: bright extensive 63; 8: water.

Fig. 2 is sensitivity test figure, and from 1 to 6 template copy number is followed successively by 10000; 2000; 500; 100; 50; 0.

Embodiment

Detailed description below by embodiment is further illustrated the present invention, but is not limitation of the present invention, only does example explanation.

The experimental technique of unreceipted actual conditions in embodiment below, conventionally according to normal condition, " molecular cloning: laboratory manual " (New York:Cold Spring Harbor Laboratory Press such as Sambrook etc., 2001) condition described in, or the condition of advising according to instrument or reagent manufacturer.

embodiment mono-: the design of primer and screening

First, design of primers and screening: during RPA design of primers, conventionally need to consider following factor: (1) GC content is at 40%-60%; (2) avoid primer inside to occur secondary structure as far as possible; (3) avoid primer to duplicate sequence.RPA requires, for 30-35nt, need to from target sequence two ends, design multipair primer to be optimized in experiment to primer length, screening, and the replacement of indivedual bases or increase and decrease all can produce material impact to experimental result.In this experiment, according to section rich No. 6 transformant distinguished sequences (GenBank No. HM124448), design a probe, in the both sides of probe, designed 4 upstream primers and 5 downstream primers.500 rich No. 6 genomic dnas of copy section of take carry out fluorescent screening to different primers combination as template, finally select the pair for amplification departure time is short and fluorescent signal is strong primer for RPA fluoroscopic examination, primer and probe sequence are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3(table 1).

Table 1

Note: FAM: luminophore; DSpacer: abasic site; BHQ1: quenching group; Phosphate: phosphate group

embodiment bis-: with the primer pair section of design, detect for rich No. 6

1. experiment material

(1) vegetable material

Transgenic paddy rice section rich No. 6 (blades), the rich No. 6 seed powder of transgenic paddy rice section (content 5%), the rich No. 6 seed powder of transgenic paddy rice section (content 1%), transgenic paddy rice Kemingdao 1(blade), transgenic paddy rice TT51-1(blade), transgenic paddy rice resists excellent 97(blade), the bright extensive 63(blade of non-transgenic paddy rice).

(2) enzyme and reagent

Molecular biology reagent, TwistAmp DNA Amplification Exo Kits is purchased from TwistDX company, and other biochemical reagents are import packing or domestic analytical pure.Primer is synthetic by Beijing Sheng Gong Bioisystech Co., Ltd.

(3) laboratory apparatus

DNA process instrumentation: low-temperature mixed ball milling instrument MM400(Retsch)

Fluorescence detector: RPA augmentation detection instrument (Twista) or quantitative real time PCR Instrument.

Other Instruments comprises: thermostat water bath, electronic balance, whizzer, vortex instrument, pure water instrument, constant incubator etc.

2. experimental technique and process

(1) extraction of oryza sativa genomic dna

Get young leaflet tablet or seed powder as DNA extraction material, according to TianGen Plant Genomic DNA Kit(Cat#DP-305) operational manual of test kit, carries out the extraction of rice total dna.

(2) DNA concentration and purity testing

Use NanoDrop 1000 spectrophotometers (Thermo Scientific) to measure purity and the concentration of DNA, and regulate DNA concentration to 50ng/ μ L with deionization distilled water.

(3) primer amplification

The transformant distinguished sequence inserting between DNA and plant genome DNA for PRA method amplification external source in the present embodiment is identified rich No. 6 of transgenic paddy rice section, and template concentrations is 50ng/ μ L.

RPA amplification system is: total system 50 μ L, in the 0.2mL TwistAmp Exo reaction tubes that contains lyophozyme powder, add rehydration damping fluid 29.5 μ L, magnesium acetate solution 2.5 μ L(280mmol/L), each 2 μ L(10 μ mol/L of primer), probe 0.5 μ L(10 μ mol/L), template DNA 50ng, residue water is supplied;

Primer amplification program: 39 degrees Celsius of reactions of RPA augmentation detection instrument 20 minutes.

3. experimental result

According to forward primer and the reverse primer of the design of transformant distinguished sequence, be SEQ ID NO.1, SEQ ID NO.2, to bright extensive 63, extensive No. 1 of China, Kemingdao 1, rich No. 6 oryza sativa genomic dnas of Kang You97He section carry out RPA fluoroscopic examination, can identify rapidly and accurately transgenic paddy rice section rich No. 6, wherein in containing the sample of rich No. 6 of transgenic paddy rice section, there is obvious amplification curve, but not all there is no amplification curve (Fig. 1) in the materials such as extensive No. 1 of transgenic paddy rice bright extensive 63 and transgenic paddy rice China.Template is diluted with water to 10000,2000,500,100,50 copies, result shows that the method can detect the target molecules (Fig. 2) of 100 copies.Explanation identifies that with the primer of the present invention's design transgenic paddy rice section has higher sensitivity and accuracy rich No. 6, and easy and simple to handle.

<110> Biological Technology institute, Chinese Academy of Agricultural Sciences

<120>application RPA technology is identified rich No. 6 strain specificities of transgenic paddy rice section

<160>?3

<210>?1

<211>?35

<212>?DNA

<400>?1

CAGATTGTCG?TTTCCCGCCT?TCAGTTTAAA?CTATC

<210>?2

<211>?35

<212>?DNA

<400>?2

TTCCATGTTT?TCTGTAGGGT?GGTCTTGGTG?GTGCC

<210>?3

<211>?41

<212>?DNA

<400>?3

AAAGATCAGG?ATTTGGGAAG?GGCGATC

GGCGAGGCAC?ATAT

Claims

1. one kind for identifying primer and the probe combinations of rich No. 6 of transgenic pest-resistant rice section by recombinase polysaccharase isothermal amplification technique, it is characterized in that: its forward primer sequence is as shown in SEQ ID No.1, reverse primer sequence is as shown in SEQ ID No.2, and probe sequence is as shown in SEQ ID No.3.

2. a test kit of identifying rich No. 6 of transgenic pest-resistant rice section, is characterized in that: this test kit comprises primer claimed in claim 1 and probe combinations.

3. a method of identifying rich No. 6 of transgenic pest-resistant rice section by recombinase polysaccharase isothermal amplification technique, it is characterized in that: extract the DNA of testing sample as template, utilize the primer described in claim 1 and probe combinations is carried out rapid amplifying and real-time fluorescence detects, if obtain obvious amplification curve, prove that institute's sample product contain rich No. 6 compositions of transgenic pest-resistant rice section.

4. method as claimed in claim 3, is characterized in that:

To adding concentration in 50 μ L amplification systems of RPA amplification kit, be each 2 μ L of 10 μ mol/L primers, concentration is 10 μ mol/L probe 0.5 μ L, template DNA 50ng;

Amplification program is: RPA augmentation detection instrument or quantitative real time PCR Instrument were in 39 degrees Celsius of reactions 20 minutes;

Carry out real-time fluorescence detection simultaneously.